TRH, a simple tripeptide synthesized in the hypothalamus, operates in the anterior pituitary to control levels of TSH and prolactin. The same peptide is found in many other tissues and appears to be involved in a wide variety of physiological activities. Elucidation of mechanism of action, identification of critical features of the molecule, separation of its multiple activities through analog design, and stabilization ot enzymatic degradation have been elusive goals for 30 years. By modification of the imidazole ring of histidine, the second amino acid in the sequence, we have successfully separated cardiovascular from pituitary activities, while replacement of histidine by valine leads to retention of CNS, but loss of both pituitary and CVS activities. Isolation and cloning of the pituitary receptor, as well as replacement by mutation of predicted key amino acids, have permitted Dr. M. Gershengorn to formulate the first model of a peptide-receptor complex which can be subjected to experimental verification. Since the first residue, pyroglutamic acid, is known to be responsible for at least half the peptide's binding energy, its role was first investigated. Removal of the carbonyl group of the 5-membered ring (replacement by proline) results in a loss of 100,000 in binding capacity. An equivalent loss is observed on replacement of tyrosine-106 in helix 7 of the seven- transmembrane-spanning, guanine-binding receptor protein by phenylalanine. It seems quite evident, therefore, that a strong h-bond exists between the OH of TYR-106 and the ring carbonyl group. Parallel manipulations show that the ring nitrogen forms a critical H-bond to asparagine-110 of helix 3. We now find that retention of the carbonyl, but replacement of the ring nitrogen by carbon, provides an analog whose binding capacity and signal-transducing ability are only slightly less than those of TRH itself. Hence, the H-bond to TYR-106 is considerably more important than that to ASN-110. These findings now permit us to delineate more accurately the stereochemistry of the binding region and to design efficient irreversible ligands. Irreversible ligands may permit us to show the existence of different conformations of TRH.